A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. Four DNaseI hypersensitive sites, HSI, II, III, and V located between 14.5 to 32 kb 5' to the pituitary hGH-N gene promoter, mark the determinants of this hGH LCR. These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI and HSII, at -14.5 to -15.5, are specific to pituitary chromatin and constitute the primary determinants of hGH-N transgene expression in somatotropes. The core elements of HSI, comprising an array of binding sites for the pituitary-enriched transfactor Pit-1, integrate multiple sets of epigenetic regulatory modifications in the process of hGH-N activation. These HSI-dependent activities include: 1) establishing of a 32kb domain of core histone hyperacetylation encompassing the hGH LCR and extending to the hGH-N promoter, 2) generation of a robust PolII 'domain of transcription' within the LCR and adjacent CD79b gene that is necessary for full levels of hGH- N expression, and 3) organization of higher-order chromatin structure that juxtaposes the LCR/CD79b domain of transcription and the hGH-N promoter during the process of hGH-N transcriptional enhancement. Remarkably, the powerful chromatin-based activities of the Pit-1 array at HSI can be functionally distinguished from the activity of the Pit-1 array at the hGH-N promoter. The HSI Pit-1 specificity is mediated, in part, by its unique binding site sequence. HSI activity appears to act in synergy with the adjacent HSII. HSII may be responsible for augmenting HSI activity via extension of epigenetic modifications or by enhancing PolII occupancy and non-coding transcription within the LCR. The role of the LCR in the activation and enhancement of hGH-N expression may be followed by an equally important role in maintaining high levels of hGH-N expression throughout adult life. In this proposal, individual components of LCR action will be characterized using a combination of in vitro, cell-based, and mouse transgenic methodologies. The temporal order of LCR-dependent alterations at the hGH locus will be determined in a novel set of tissue culture lines that are arrested at defined stages of somatotrope differentiation. A naturally occurring mutation of the human Pit-1 protein that selectively blocks hGH-N expression will be used to further extend our understanding of the basis for the specificity of Pit-1 action at the hGH LCR. The Specific Aims of this proposal focus on these major aspects of hGH LCR function with the ultimate goal of extending our understanding of hGH-N activation and expression and in generalizing our findings to pathways and mechanisms of long-range transcriptional activation in the eukaryotic genome. These findings will eventually be correlated with an array of pathologic defects in hGH expression, both inherited and acquired, and with physiologic alterations in hGH expression that occur in response to environmental stress and ageing.